Sequestration of the Aβ Peptide Prevents Toxicity and Promotes Degradation In Vivo

Abstract

Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Aβ) peptide by using a small engineered binding protein (Z_Aβ3) that binds with nanomolar affinity to Aβ, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z_Aβ3 in the brains of Drosophila melanogaster expressing either Aβ₄₂ or the aggressive familial associated E22G variant of Aβ₄₂ abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Aβ binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Aβ aggregation and reveal that Z_Aβ3 not only inhibits the initial association of Aβ monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation. Alzheimer's disease is thought to be a result of neuronal damage caused by toxic aggregated forms of the Aβ peptide in the brain. There is no cure and existing treatments are ineffective in reversing or preventing disease progression. Here we describe a novel strategy that makes use of an engineered “Affibody” protein to study the disease and potentially combat its underlying causes. The Affibody occludes the aggregation-prone regions of Aβ peptides, preventing their aggregation into toxic forms, and it also acts to dissolve pre-formed Aβ aggregates. It is functional in vivo, as its co-expression with Aβ peptides in transgenic fruit flies prevents the neuronal damage and premature death that result from expression of Aβ peptides alone. Moreover, we show that the origin of this protection is the enhanced clearance of Aβ peptides from the brain. These findings open up new opportunities for using engineered binding proteins to probe the origins of Alzheimer's disease and potentially to develop a new class of therapeutic agents.